Association of cytosol hemoglobin with the membrane in intact erythrocytes.
The problem of demonstrating hemoglobin binding to the erythrocyte membrane in intact cells was approached by observing the quenching of fluorescent membrane probes by hemoglobin as a function of pH. This quenching was studied by measuring fluorescence intensities and decay rates of membrane-bound donors by both right-angle and front-face fluorometry. The donors included 4,4'-bis(isothiocyano)-2,2'-stilbene disulfonate (DIDS) bound to the band 3 protein and 2-, 6-, and 12-(9-anthroyloxy(stearic acid (2-AS, 6-AS, and 12-AS) in the lipid portion of the membrane. The probe fluorescence is quenched progressively as the intracellular pH is decreased from 7.2 to 5.9 and does not depend on the oxygenation state. Since the fluorescence characteristic of the DIDS and x-AS probes in ghosts are independent of pH over this range, this quenching is due to the greater proximity of the donors and hemoglobin. Because hemoglobin binding to the band 3 protein in ghosts has previously been shown under conditions of low ionic strength and hemoglobin concentration, the present results were analyzed by a model in which a hemoglobin molecule is bound or in close proximity to a band 3 protein at pH 6. By using resonance energy transfer theory, we found the distance from DIDS to the bound hemoglobin to be approximately 4 A, which is within the range of distances measured between the DIDS binding site and the band 3 protein cytoplasmic sulfhydryl groups. Furthermore, the pH-dependent fluorescence quenching of 12-AS was stronger than that of 2-AS, showing that a cytoplasmic acceptor is involved and that the average distance between hemoglobin and these probes was greater than that for DIDS.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives; D001457:Anion Exchange Protein 1, Erythrocyte; D001798:Blood Proteins / Q000378:metabolism*; D002245:Carbon Dioxide / Q000097:blood; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D001457:Anion Exchange Protein 1, Erythrocyte; D001798:Blood Proteins; D006454:Hemoglobins; D002245:Carbon Dioxide; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid